Cells continuously secrete a large number of microvesicles, nanovesicles, macromolecular complexes, and small molecules into the extracellular space. Exosomes are small secreted vesicles (typically about 30-150 nm) which may contain or have present in their membrane nucleic acid, protein, or other biomolecules and may serve as carriers of this cargo between diverse locations in the body (Mittelbrunn & Sanchez-Madrid, Nature Reviews 13 (2012) 328-335; Thery et al. Nat. Rev. Immunol. 2 (2002) 569-579; Valadi et al. Nat. Cell. Biol. 9 (2007) 654-659). Exosomes are secreted by all types of cells in culture, and also found in abundance in body fluids including blood, saliva, urine, and breast milk (Kosaka et al., Silence 3 (2010) 1-7; Mitchell et al., PNAS 105 (2008) 10513-10518; Palanisamy et al., PLoS One 5 (2010) e8577).
Currently, the control of exosome formation, the makeup of the “cargo”, biological pathways and resulting functions are incompletely understood. One of their most intriguing roles is intercellular communication. Exosomes are thought to function as messengers, delivering various effector or signaling macromolecules between cells.
The accepted protocol for isolation of exosomes includes ultracentrifugation (Thery et al., Curr. Protoc. Cell. Biol., Chapter 3, Unit 3: 22 (2006)), often in combination with sucrose density gradients or sucrose cushions to float the relatively low-density exosomes. Isolation of exosomes by sequential differential centrifugations is complicated by the possibility of overlapping size distributions with other microvesicles or macromolecular complexes. Furthermore, centrifugation may provide insufficient means to separate vesicles based on their sizes. However, sequential centrifugations, when combined with sucrose gradient ultracentrifugation, can provide high enrichment of exosomes.
Isolation of exosomes based on size, using alternatives to the ultracentrifugation routes, is another option. Successful purification of exosomes using ultrafiltration procedures that are less time consuming than ultracentrifugation, and do not require use of special equipment have been reported (Cheruvanky et al., J. Physiol. Renal Physiol. 292 (2007) 1657-1661.) Similarly, a commercial kit is available (ExomiR, Bioo Scientific) which allows removal of cells, platelets and cellular debris on one microfilter and capturing of vesicles bigger than 30 nm on a second microfilter using positive pressure to drive the fluid. For this process, the exosomes are not recovered, their RNA content is directly extracted off the material caught on the second microfilter, which can then be used for PCR analysis. HPLC-based protocols could potentially allow one to obtain highly pure exosomes, though these processes require dedicated equipment and are difficult to scale up. A significant problem is that both blood and cell culture media contain large numbers of nanoparticles (some non-vesicular) in the same size range as exosomes. For example, Wang et al. (Nucleic Acids Res. 38 (2010) 7248-7259.) found that large number of miRNAs are contained within extracellular protein complexes rather than exosomes. As a consequence, the above methods are best described as allowing one to obtain exosome-enriched samples, rather than pure exosomes.
Volume-excluding polymers such as PEGs can sometimes be used for precipitation of viruses and other small particles (Yamamoto et al. Virology 40 (1970) 734-744; Adams, J. Gen. Virol. 20 (1973) 391-394; Lewis et al., Applied and Environmental Microbiol. 4 (1988) 1983-1988). We have unexpectedly found that despite exosomes being noticeably less dense than viruses due to the lack of a protein coat and variable, though probably lower (for their size), nucleic acid content, volume-excluding polymers are capable of differentially precipitating exosomes thereby allowing exosome isolation by low-speed (benchtop) centrifugation or filtration.